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Summary

The FLC gene encodes a MADS box repressor of flowering that is the main cause of the late-flowering phenotype of many Arabidopsis ecotypes. Expression of FLC is repressed by vernalization; maintenance of this repression is associated with the deposition of histone 3 K27 trimethylation (H3K27me3) at the FLC locus. However, whether this increased H3K27me3 is a consequence of reduced FLC transcription or the cause of transcriptional repression is not well defined. In this study we investigate the effect of changes in transcription rate on the abundance of H3K27me3 in the FLC gene body, a chromatin region that includes sequences required to maintain FLC repression following vernalization. We show that H3K27me3 is inversely correlated with transcription across the FLC gene body in a range of ecotypes and mutants with different flowering times. We demonstrate that the FLC gene body becomes marked with H3K27me3 in the absence of transcription. When transcription of the gene body is directed by an inducible promoter, H3K27me3 is removed following activation of transcription and H3K27me3 is added after transcription is decreased. The rate of addition of H3K27me3 to the FLC transgene following inactivation of transcription is similar to that observed in the FLC gene body following vernalization. Our data suggest that reduction of FLC transcription during vernalization leads to an increase of H3K27me3 levels in the FLC gene body that in turn maintains FLC repression.

Figure S3. Kinetics of H3K27me3 addition after vernalization and following decreased transcription of tFLC are similar. H3K27me3 ChIP at amplicon 7 (normalised to FUS3 H3K27me3) plotted against time after removal from dexamethasone for flc-2 pOp6:tFLC plants and time after removal from cold for the endogenous FLC gene in ColFRI. Linear regression lines and R2 co-efficients are shown on the plot.

Table S1. Oligonucleotide sequences. Primer sets 3a and 10a amplify longer (approximately 200 nt) products than 3b and 10b (approximately 100 nt). The ‘a’ and ‘b’ primer sets gave the same relative differences between samples when tested on the same ChIP DNA. Primer sets 5a and 5b amplify at a similar location, 5a gives non-specific products in the absence of the target region in flc-2, but is specific in the presence of FLC, 5b does not amplify from flc-2.

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